The Prostaglandin Synthase 2 / cyclooxygenase 2 ( PTGS 2 / COX 2 ) rs 5277 Polymorphism Does not Influence Risk of Colorectal Cancer in an Iranian Population

Inflammation plays a key role in the development of colorectal cancer. Several studies revealed that inflammatory disease of the colon, such as Crohn’s disease and Ulcerative colitis, substantially increase the risk of colorectal cancer (Lennard-Jones et al., 1977; Lashner et al., 1989; Rhodes and Campbell, 2002). Prostaglandins are molecules of particular interest in the inflammatory response. The cyclooxygenase (COX) enzymes, also known as prostaglandin H2 synthase (PGHS) or prostaglandin-endoperoxide synthase (PTGS), are key enzymes in the production of prostaglandins. Currently, two isoenzymes of COX have been identified; COX1 and COX2. COX-1 (PTGS1) which is considered to be a constitutive enzyme expressed in most cell types and normal tissues (Dubois et al., 1998; Vane et al., 1998). Conversely, the COX2 (PTGS2) isoform, undetectable under normal physiological conditions, is


Introduction
Inflammation plays a key role in the development of colorectal cancer.Several studies revealed that inflammatory disease of the colon, such as Crohn's disease and Ulcerative colitis, substantially increase the risk of colorectal cancer (Lennard-Jones et al., 1977;Lashner et al., 1989;Rhodes and Campbell, 2002).
Prostaglandins are molecules of particular interest in the inflammatory response.The cyclooxygenase (COX) enzymes, also known as prostaglandin H 2 synthase (PGHS) or prostaglandin-endoperoxide synthase (PTGS), are key enzymes in the production of prostaglandins.Currently, two isoenzymes of COX have been identified; COX1 and COX2.COX-1 (PTGS1) which is considered to be a constitutive enzyme expressed in most cell types and normal tissues (Dubois et al., 1998;Vane et al., 1998).Conversely, the COX2 (PTGS2) isoform, undetectable under normal physiological conditions, is an inducible enzyme therefore is induced in response to cytokines, mitogens, growth factors, tumor promoters, proinflammatory stimuli and tumor development (Dubois et al., 1998;Bakhle, 2001;Cao and Prescott, 2002).Recently, researchers have demonstrated that COX-2 could have had a substantial role in the etiology of colorectal and other cancers (Brown and DuBois, 2005;Eisinger et al., 2007).
It has been indicated that overexpression of COX-2 is correlated with tumor recurrence, shorter survival, lymphatic metastases, later stage and larger tumor size and particularly with hematogenous spread of the colorectal cancer (Sheehan et al., 1999;Tomozawa et al., 2000;Zhang and Sun, 2002;Soumaoro et al., 2004).Furthermore, expression of COX-2 is thought to accommodate tumor promotion and carcinogenesis through stimulation of cell proliferation, inhibition of apoptosis, and of angiogenesis and invasiveness (Cao and Prescott, 2002;Kanaoka et al., 2007).
We have selected gene polymorphism rs5277 (3050) located in the coding region of exon 3 on the basis of reported functional and biological relevance.Previous studies have shown that the selected polymorphism are the more prevalent and has biological relevance in colorectal cancer (Barry et al., 2009;Pereira et al., 2009).Besides the COX2.3050polymorphism is one of the most prevalent in the Caucasian population and it has been studied in several pathologic disorders, and associations with disease have been found (Campa et al., 2004;Cheng et al., 2007;Cox et al., 2007;Lee et al., 2007), however, the functional impact of this polymorphism on COX-2 activity still remains unknown.There is one hypothesis that this polymorphism itself or with another linked marker may have biological effects on COX-2 activity (Xaubet et al., 2010).
The aim of this study was to assess the association between single nucleotide polymorphism rs5277 of COX-2 gene involved in the Prostaglandins pathway and the occurrence of colorectal cancer in an Iranian population.

Study population and sample collection
This case-control study was conducted to assess risk factors of colorectal cancer.Cases were 167 patients with a histologically confirmed diagnosis of colorectal cancer attending the Taleghani Hospital in Tehran, Iran, between 2007 and2011. 197 subjects without any colorectal tumors or other tumors were evaluated as control group.This study was confirmed by the Ethics Committee of the Research Center for Gastroenterology and Liver diseases, Shahid Beheshti University of Medical Sciences.

DNA extraction
Peripheral blood samples of the both groups (cases and controls) were processed for the DNA extraction, immediately after their collection.DNA was isolated by standard methods using proteinase K digestion, phenol chloroform extraction and ethanol precipitation and stored at 4˚C.DNA concentration was determined by using Nanodrop (Thermo Scientific NanoDrop 1000 Spectrophotometer/USA).
The PCR profile consisted of an initial denaturation of 95ºC for 5 min, followed by 30 cycles of 95ºC for 45 s, 60.5ºC for 40 s, and 72ºC for 40 s with final extension of 72ºC for 5 min.PCR products (10µl) were incubated with 10U of restriction enzyme HincII at 37ºC overnight and analyzed by 2.5% agarose gel electrophoresis, and visualized by ethidium bromide staining.The 306G>C genotypes that could be detected were: 306CC (511 bp fragment), 306GC (511+424+87 bp fragments), and 306GG (424 + 87 bp fragments).The results of the RFLP (restriction fragment length polymorphism) analysis were confirmed by randomly selected samples for direct sequencing (Figure 1).

Statistical analysis
Data were analyzed using SPSS 13.The X 2 or Fisher's exact tests were applied to determine differences in genotype/allele frequencies.The confounding effect of age and gender was adjusted using conditional logistic regression.The p value <0.05 was considered as significant.Data were expressed as mean±SD or frequency (%).Logistic regression was used to calculate odds ratio (OR) and 95% confidence interval (CI) for the association between genotypes and sporadic CRC.The observed genotype frequencies were tested by X 2 test for deviation from the Hardy-Weinberg equilibrium.

Results
Selected characteristics of cases and controls in the studied population are displayed in Table 1.
One hundred and sixty seven patients with the diagnosis of CRC with mean age of 57.22±13.195years, including 94 males (56.3%) and 73 females (43.7%) were studied, the control group consisted of 197 noncancer subjects with mean age of 44.43±15.639years, including 84 males (42.6%) and 113 females (57.4%).Of the patients, 114 had colon cancer and 53 had rectal cancer.BMI was 25.49±4.24for cases and 25.05±3.76for controls.Smoking status was not statistically different (p=0.281) between the two group.Approximately more NSAID consumers (13.7% vs 4.2%) were found among controls compared to sporadic colorectal cancer cases.
To remove the effect of confounder variables, the logistic regression method was used for adjustment of data.There was no significant deviation in the genotype frequency of rs5277 polymorphism from the Hardy-Weinberg equilibrium (p>0.05).
There was no significant difference in the distribution of COX-2 gene rs5277 polymorphism genotype and the allelic form, when CRC patients were compared with the healthy control group (p: 0.867).The distribution of genotype and allele frequency among the healthy controls and the patients are revealed in Table 2.In the overall studied samples, the rs5277 polymorphism was not significant associated with a CRC risk.
The potential association of genotype distribution of the COX-2 polymorphism with tumor localization and metastasis was studied.No association between the rs5277.

Discussion
Influence of genetic alterations regarding the increase or decrease of the risk of cancer development has been demonstrated by various investigations (Loktionov, 2004;Park et al., 2006;Mahmoudi et al., 2010;Lubbe et al., 2011).Recent studies has been reported a number of associations between a variety of SNPs in COX-2 and colorectal cancer (Lin et al., 2002;Cox et al., 2004;Siezen et al., 2005;Ulrich et al., 2005).Our previous study has reported association between rs20417 polymorphism in Cox-2 and sporadic colorectal cancer in Iranian population (Khorshidi et al., 2013).Accordingly, These results accentuate the significance of this gene and the pro-inflammatory AA-pathway in the development of colorectal cancer (Siezen et al., 2006).
The present study was designed to determine the probable association between one of the polymorphisms of COX-2 gene and the risk of CRC among Iranian population.
To our knowledge, this is the first study on association between rs5277 polymorphism in COX-2 and sporadic colorectal cancer in Iranian population.The frequencies of the genotypes in our patients with colorectal cancer were 82.0%GG, 16.8% GC and 1.2% CC, respectively.No significant correlation is established between both allele and genotype frequencies of PTGS2 rs5277 polymorphism and sporadic CRC risk in population under study (p value=0.867).However, the findings of the current study do not support some of the previous research which was declared a significant association between this polymorphism and CRC.For instance, in one study Barry et al. (2009) reported a statistically significant increased risk for adenoma recurrence of 49% for the rs5277 CC genotype, based on this study demonstrated the pattern of rs5277 was indicative of a recessive inheritance mode because heterozygotes did not have an increased risk (Barry et al., 2009).In the pooled analysis showed V102V COX-2 polymorphism influenced the development of colorectal cancers in early stages of carcinogenesis (Pereira et al., 2009) furthermore there was a non-significant trends toward a reduced adenoma risk based on two previous studies (Gunter et al., 2006;Siezen et al., 2006).Yu et al. (2010) in a report related that rs5277 attained a marginal significant in breast cancer (Yu et al., 2010).GC genotype of SNP V102V has been shown to be inversely associated with risk of colorectal cancer (Siezen et al., 2006).Besides, in an aspirin trial of colorectal cancer, rs5277 polymorphism was associated with increased cancer recurrence (Menter et al., 2010).Nonetheless, in our study, genotypes stratification by aspirin or NSAID intake and smoking status did not show any altered effect of rs5277 polymorphism on the development of sporadic colorectal cancer.Therefore, in our population this polymorphism is not influenced by aspirin or NSAID use and smoking.Moreover, one study was reported that no significant association was found between rs5277 polymorphism and cervical cancer risk in the Korean population (Lee et al., 2007).Also, our finding is supported by a study in United State which is reported no statistically significant association between rs5277 and colon cancer risk (Thompson et al., 2009).As a final point, some of the previous studies have shown rs5277 polymorphism is not associated with risks of Gastric cancer and (Hou et al., 2007), Prostate cancer (Danforth et al., 2008).Apparently, further investigations are required to clarify these conflicting findings.
In conclusion, this preparatory study was the first report of rs5277 COX-2 gene polymorphism among colorectal cancer patients from Iran.Overall, there was no association between COX-2 rs5277 polymorphism and the risk of sporadic CRC in Iranian population.Further studies to replicate this finding in different populations are needed to validate this result.

Table 2 . Allele and Genotype Distribution of Studied SNP among CRC Patients and Healthy
*Adjusted for confounder variables